Journal: Bioengineering & translational medicine

Article Title: 3D bioprinting of an implantable xeno-free vascularized human skin graft.

doi: 10.1002/btm2.10324

Figure Lengend Snippet: FIGURE 5 Phenotyping characterization of human keratinocytes under xeno-free conditions. (a) Live phasecontrast microscopy images of keratinocytes after isolation from the epidermis of donor foreskin and at the confluency state. (b) The cumulative population doublings of keratinocytes cultured under xeno-free conditions is comparable to KGM-Gold medium. (c) Flow cytometry analysis confirmed the expression of integrins α2β1, α5β1, α6, α3, and β4, but not αvβ3. (d) Confocal microscopy exhibiting CK14, CK10, junctional ZO-1, and intracellular occludin staining. Scale bar = 100 μm. Representative of three independent donors

Article Snippet: ECs, FBs, PCs, and KCs cultured under xeno-free conditions were analyzed for surface and intracellular markers expression by flow cytometry and immunofluorescence microscopy using antibodies against: CD31 (WM59; Biolegend), CD45 (2D1; Biolegend), ZO-1 (sc-33725; Santa Cruz), VE-cadherin (sc-6458; Santa Cruz), vWF (ab201336; abcam) claudin-5 (34-1600; Invitrogen), PDGFR-α (16A1; Biolegend), PDGFR-β (18A2; Biolegend), CD90 (5E10; Biolegend), NG2 (9.2.27; Invitrogen), a-SMA (1A4; Invitrogen), FAP (AF3715; Novus Biologics), integrins α2β1 (ab24697; abcam), α5β1 (NBP2-52680; Novus Biologics), αvβ3 (sc-7312; Santa Cruz), α6 (MAB13501; R&D systems), α3 (MAB1345; R&D systems), β4 (NBP1-43369), CK14 (LL002; Novus Biologics), CK10 (EP1607IHCY, abcam), and occludin.

Techniques: Microscopy, Isolation, Cell Culture, Flow Cytometry, Expressing, Confocal Microscopy, Staining

Journal: Nature Communications

Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side

doi: 10.1038/s41467-019-13877-w

Figure Lengend Snippet: a Schematic of a cell interacting with a tethered virus particle from its ventral side (not to scale). PEG-passivated glass coverslips were decorated with cRGD ligands (gray) for integrin-specific cell adhesion and tension sensors consisting of gold nanoparticles (AuNPs, yellow) and Alexa647-labeled titin I27 domain (blue). Alkyne-modified reoviruses (magenta) were tethered via click chemistry (CuAAC). Fluorescence of the tension probes is quenched by the AuNP via nanometal surface energy transfer (NSET) and increases upon unfolding. b SEM images of a BSC1 cell on an array of tension sensors (asterisk) onto which single virus particles are bound (arrowhead). Scale bars, 5 μm (overview), 1 μm (upper), 200 nm (lower panel). c Maximum projections of TIRF images over 10 min of titin-based tension sensors (green) and fluorescently labeled reoviruses (magenta) and merge with the cell outline (see Supplementary Movie ). Arrowheads indicate signals of opened tension sensors at virus sites. Hollow arrowhead points towards a nonspecific tension signal. Scale bar, 10 μm. Fluorescence intensity traces were analyzed at sites of viruses (white circle) or at random spots not co-localizing with viruses after subtraction of the local background signal (gray annulus). Reovirus signal decays over time due to photobleaching of the sparsely Alexa568-labeled virus. d Rate k unfold of tension sensors with characteristic increase in fluorescence representing the tension sensor unfolding at sites of virus immobilization/at random spots under the cells and in a control region outside of cells during 10 min, 1 h post seeding cells ( n = 10, 11, 12, 13 cells or control regions, with a total number of 114 events out of 1073 virus particles, 88 non-specific events out of 2549 random spots underneath the cells, 40 events per 1228 viruses and 80 non-specific events out of 2713 random spots in the control region, respectively, three technical replicates, box-plots represent median ± 95% CI with whiskers to the min and max values, **** P < 0.0001, one-way ANOVA with post hoc Tukey). Source data are provided as a source data file.

Article Snippet: To block β 1 integrins, 10 5 HeLa cells were incubated in 200 μl serum-free media equipped with human integrin β 1 (CD29) blocking antibody clone P5D2 (#MAB117781, R&D systems, USA) at 10 μg ml −1 final concentration for one hour before seeding.

Techniques: Virus, Labeling, Modification, Fluorescence, Control

Journal: Nature Communications

Article Title: Forces during cellular uptake of viruses and nanoparticles at the ventral side

doi: 10.1038/s41467-019-13877-w

Figure Lengend Snippet: a Schematic of a cell tearing off a biotin-neutravidin bound nanoparticle (AuNP) either only passivated with PEG (1) or additionally functionalized with cRGD (2) or integrin α 5 β 1 selective ligands (3). b Confocal images of a HeLa cell (transmission) spreading and tearing off StarRed-fluorescently labeled AuNPs (magenta) from the surface at t = t 0 + 70 min with ROIs 1 h post seeding. Scale bars, 10 µm. c Relative number of particles in the ROI under the cells or in a control region outside the cells over time. Data are represented as mean ± s.e.m. and fitted with two-phase decay functions. ( n = 3 control regions or 19, 24, 27 cells on AuNPs (1), (2) and (3), respectively, three technical replicates, data are represented as mean ± s.e.m. and fitted with an exponential decay function). d Projected cell area normalized to t 0 of cells spreading on the matrix-mimetic surfaces decorated with AuNPs 1–3 over time with exponential fits. e For the particles being actively removed by the HeLa cells, the fraction a and the off rate k off are shown as obtained by fitting from the two-phase-decay of particles in the ROI as presented in ( c ). (Box-plots represent median ± 95% CI with whiskers to the min and max values, ** P < 0.001, **** P < 0.0001; one-way ANOVA with post hoc Tukey). Source data are provided as a source data file.

Article Snippet: To block β 1 integrins, 10 5 HeLa cells were incubated in 200 μl serum-free media equipped with human integrin β 1 (CD29) blocking antibody clone P5D2 (#MAB117781, R&D systems, USA) at 10 μg ml −1 final concentration for one hour before seeding.

Techniques: Transmission Assay, Labeling, Control

Journal: Oncology Letters

Article Title: Extracellular matrix differences in glioblastoma patients with different prognoses

doi: 10.3892/ol.2018.9649

Figure Lengend Snippet: Primary antibodies used for immunohistochemical staining.

Article Snippet: Integrin beta 1 chain , RD-MAB1778-SP , RnD Systems (Minneapolis MN, USA , 1:250 , Human tonsil.

Techniques: Immunohistochemical staining, Staining, Positive Control

Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

Figure Lengend Snippet: ( A, B ) The human ( A ) and mouse ( B ) Osteolectin proteins contain RGD and LDT sequences. ( C ) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. ( D, E ) RNA-seq analysis of integrin α ( D ) and β ( E ) subunits in PDGFRα + CD45 - Ter119 - CD31 - bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression . ( F ) RNA-seq analysis of Itga1 , Itga6 , Itga11 , and Itgav in PDGFRα + CD45 - Ter119 - CD31 - bone marrow stromal cells, VE-Cadherin + bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). ( G ) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in . ( H ) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin β1 and integrin α11 with Flag-tagged Osteolectin (results are representative of two independent experiments). ( I ) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin α 11 β 1 and α 10 β 1 , but not to other integrins (n = 3 independent experiments). ( J ) Integrin α11β1 bound Osteolectin and recombinant human Pro-Collagen 1α (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). ( K ) Osteolectin, but not Pro-Collagen 1α, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). ( L ) 200 nM RGDS peptide inhibited the binding of integrin α11β1 to recombinant human Osteolectin. ( M ) 100 μM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way ( G ) or two-way ANOVAs with Dunnett’s multiple comparisons tests ( K ) or Tukey’s multiple comparisons tests ( M ). 10.7554/eLife.42274.004 Figure 1—source data 1. Data for .

Article Snippet: Peptide, recombinant protein , recombiant Integrin α4β1 protein , R and D Systems , 5668-A4 , .

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Isolation, Immunoprecipitation, Recombinant, Binding Assay, Standard Deviation

Journal: eLife

Article Title: Integrin alpha11 is an Osteolectin receptor and is required for the maintenance of adult skeletal bone mass

doi: 10.7554/eLife.42274

Figure Lengend Snippet:

Article Snippet: Peptide, recombinant protein , recombiant Integrin α4β1 protein , R and D Systems , 5668-A4 , .

Techniques: Recombinant, Diagnostic Assay, Cell Culture, Protease Inhibitor, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Fractionation